After one centrifugation (picture by MK); the picture on the right is borrowed from: http://www.malariasite.com/malaria/QBC.htm 

BD reagent droppers (Eng.): http://www.bd.com/ds/productCenter/261182.asp 
(Dansk: http://www.bd.com/resource.aspx?IDX=19360)

QUOTE: "Acridine orange binds to nucleic acids of cells and bacteria [DNA, RNA]. When viewed under UV light, single-stranded DNA and RNA fluoresce orange, whereas double-stranded DNA appears green. At low pH (3.5 – 4.0), bacteria and fungi stain bright orange.
Cellular material stains pale green to yellow.2   Nuclei of activated leukocytes may stain orange, yellow or red, depending upon the degree of increased RNA production.
Erythrocytes either have no color or appear pale green."

"The literature on the discovery, development and validation of the AO [acridine orange] method for malaria diagnosis is reviewed here."
"The basic feature of fluorescent staining is that, in contrast to the mature erythrocytes that do not contain DNA or RNA, the nucleic acids of the parasite fluoresce strongly."
"After testing 30 different fluorescent dyes, Fuhrmann (1962) found an AO solution with a pH between 6 and 7 and a concentration of 1: 10,000 to be the most promising dye for the staining of thin bloodsmears containing Babesia canis, P. berghei or P. cathemerium. ... shortly thereafter, excellent results were reported for the AO staining not only of thin smears containing P. berghei or P. vivax (Ambroise-Thomas et al., 1965) but also of thick smears containing various Plasmodium species (Sodeman, 1970). In the latter study, the thick smear was made slightly thinner than was then customary and dehaemoglobinized during staining with a 0.01% AO solution of pH 5.4 (Sodeman, 1970). Commonly, AO stain is applied to a thin smear only after the smear has been dried and then fixed in methanol. However, an immediate method, in which AO solution is applied directly to an unfixed and undried blood film, has also been described (Kawamoto, 1991 a; Metzger and Nkeyi 1995)".
Fluorecens microscopy was for a long time too expensive but ... "In 1991, however, the development of a relatively cheap, interference-filter system (a multi-layered excitation filter combined with a barrier filter) designed for use with AO in a standard light microscope provided a low-cost tool for malaria diagnosis employing AO (Kawamoto, 1991 b).
A combination of centrifugation, AO staining and fluorescence microscopy was developed into the quantitative buffy coat (QBC) method. Although this technique appears to be easy to handle, sensitive and rapid, it requires costly pre-prepared tubes, a centrifuge and an ultra-violet microscope (Lema et al., 1999). It is not being discussed here in more detail."

Borrelia (and probably other spirochetes?)
stain yellow to red-orange (single stranded DNA and RNA) with Acridine Orange - examples:

Borrelia hermsii

http://wwwnc.cdc.gov/eid/images/01-0198-F2.jpg

Borrelia duttonii
Borrelia burgdorferi

stained with Acridine Orange, by Alan MacDonald from
http://www.molecularalzheimer.org

Borrelia burgdorferi - cystic form

from Brorson et al. Destruction of spirochete Borrelia burgdorferi round-body propagules (RBs) by the antibiotic Tigecycline
http://www.pnas.org/content/106/44/18656.full.pdf


from http://www.molecularalzheimer.org

Trypanosoma

http://www.finddiagnostics.org/export/sites/default/programs/hat-ond/images/Trypanosomes_40x_Acridine_orange.jpg
http://www.finddiagnostics.org/programs/hat-ond/hat/parasite_detection/fluorescence_microscopy/

Malaria

http://www.qbcdiagnostics.com/products/fm/malaria/files/malaria_appnote_pla.pdf

Another fluorochrome stain
Tuberculosis in Auramine O stain, not Acridine Orange!

http://www.zeiss.com/C12567BE00472A5C/GraphikTitelIntern/Tuberkel_fluo_big/$File/Tuberkel_fluo_big.jpg

"The QBC Malaria method is the simplest and most sensitive method for diagnosing the following diseases.

Some research references are shown here. "

  old versus new Paralens

"Fluorescent antibody (FA) technique, also known as immunofluorescence, is an excellent rapid diagnostic method. FA is easily done, sensitive, specific, and relatively inexpensive. It is extremely versatile. FA detects and identifies both etiologic agents of disease (direct FA ~DFA) and host antibodies (Indirect FA, IFA). A wide variety of infectious diseases can be rapidly diagnosed by FA. Kits and reagents for FA tests are commercially available, and many use highly specific monoclonal antibodies." ...
"Commercially available monoclonal antibodies can be used for doing indirect FA rapid tests to diagnose other infectious diseases (fluorescent anti-globulins commercially available). Some of these are: (see table on the website)" ...
"Polyclonal antibodies to these etiologic agents and others are also commercially available. Many of these are suitable for both direct and indirect FA tests."

Picture taken by Alan MacDonald (ca. 1985), borrowed with permission.
Both spirochete and cyst form and "granules" stain with Borrelia antibodies!


From Embers et al. (Full text):
Persistence of Borrelia burgdorferi in Rhesus Macaques following Antibiotic Treatment of Disseminated Infection (2012)
Figure 5. Spirochetes recovered by xenodiagnosis from animals treated in the disseminated phase of infection.
Images from direct fluorescent staining of B. burgdorferi spirochetes found in xenodiagnostic tick midgut culture (A)
or tick midgut preparation (B) from treated animals GA59 and GB56, respectively.

Note that the above Borrelia burgdorferi spirochetes do not look very spiralled
(In TBRF pictures the spirochetes often seem more coiled than B. burgdorferi).

BUT all those who claim that Borrelia burgdorferi spirochetes has so and so many coils
and is always of a certain length and thickness
- and say that what I call spirochetes do NOT look like ideal Borrelia spirochetes from pictures
- have obviously not spend years studying the morphology and
movements of live spirochetes in the microscope themselves!

Enjoy especially the amazing movies at (Youtube searching for Lymebugs videos)
- all microscopies were made by Stan Dembowsky in 1999 from his studies
of the laboratory grown spirochete Borrelia burgdorferi B31
... demonstrate:
Borrelia uncoiling itself and changing morphology at its will pretty fast
Growth by division, growth in colonies within biofilm like substance,
adults spirochete form and baby-spirochetes, blebbing, cyst form ..
Growth of Borrelia B31 on sheep blood agar, where baby-spirochetes
seem to be emerging from the surface of the red blood cells?
Stan also studied the effect of penicillin on Borrelia B31 
...

The very important Borrelia relapse pattern:

Note that spirochetes are only visible in the blood during the symptomatic episodes, but the blood was still (more) contageous also in between the relapses, when there is no visible spirochetes in the blood:
http://lymerick.net/1914-Nicolle.htm
=> CORRECT TIMING of sampling of blood specimen for microscopíc examination for spirochetes in relation to the patient relapse cycle (during attacks) is essential, if one  does not time the sample right there is a high risk of not seeing any spirochaetes!
In case the patient has a cycle that runs over 10 days, we only have 10% chance of doing it on day one in a new relapse, if one just take a random sample, and NOT time the sampling according to the relapse cycle; in case a patient has a monthly relapse pattern the chance of finding spirochetes is 1/30 i.e. 3%! 
I only have greater "luck" finding spirochetes by microscopy, than most other investigators, because I understand the relapsing pattern, and plan after it; I ask the patients to do a very detailed Excel symptom diary (OA but respect my ownership, dont remove my name) and from the relapse cycle I can deduct, when is the best time to see the patient and do sampling for direct test for spirochetes! - and can judge effect of any intervention.

A immunocompetent relapsing fever Borrelia patient will typically get from 10-30 relapses, before the disease "burn out" when the immune system achieves control over the microbial proliferation  .. probably likewise for most patients infected with Borrelia burgdorferi?
Symptom flares (attacks) are gradually diminishing in severity (relating to the amount of spirochetes that enters the blood and raise complement cascade reaction (you should measure complement split products if available) and proinflammatory cytokine storm (TNF => fever, IL-1, IL-6 ...) and late in the course, also the intervals between relapses is increasing, as long as the infection is kept under control (by immune system and/or antibiotics) then newly formed spirochetes are not allowed to reproduce themselves weekly any more ...

It is usually not mentioned in the literature that Lyme borreliosis also clinically gives a relapsing infection pattern, just like the close relatives in the relapsing fever Borreliosis group (except TBRF is perhaps more pathogenic, cause a higher TNF (fever) response than the Lyme borreliae does in the immunocompetent host?).
BUT there are some works examining this subject  ..
In very ACTIVE LATE (> 6 months post infection) Lyme borreliosis, where moving spirochetes can repeatedly be found in buffy-coat blood fraction from the patient by microscopy, but usually only during the first day of a new flare, the clinical relapse pattern is the same, weekly relapse pattern, as descibed in TBRF!
Whenever the borrelia infection is under relative control by the immune system and/or antibiotic treatment, the relapse cycle shift pattern, usually within 4-6 weeks after start on antibiotic treatment, from the previous weekly to a monthly cycle at 3-4 weeks intervals!
The monthly clinical relapse cycle was  described by dr. Joe Burrascano in his diagnostic guidelines, already since the early 1990ies, see ILADS: http://ilads.org/lyme_disease/treatment_guidelines.html

Cyclicity was also noted by Willy Burgdorfer  ...

The two relapse patterns, that are both characteristic of clinical Lyme borreliosis, are probably well explained by Brorsons laboratory culture studies, finding that it take the YOUNG cystic forms of Borrelia about 9 days versus for OLD cystic forms about 4 weeks to reproduce the spirochaete form:
http://lymerick.net/1998-Brorson.htm

The patients with VERY ACTIVE, weekly, RELAPSE pattern usually will respond to antibiotic treatment in a quite typical and gradual way, as illustrated by symptom log curves from case#24, who kept my Excel symptom diary (early version); there are 7 days in between the dates on the X-axis; arrows indicate the circa weekly clinical worsenings (6-14 days acc. to old research on RF-spirochete lifecycle done a century ago):

The monthly relapse cycle (some more intensive than others) is illustrated by curve from project patient #11 (still one week in between dates on X-axis):

More on videomicroscopy for spirochetes and the Excel symptomdiary (free download):
http://lymerick.net/MK-videomicroscopy.html 
My studies done before 2006 using direct fluorescent immunostain with borrelia specific antibodies from KPL (done as research in BRT), and primitive cheap equipment: http://lymerick.net/videomicroscopy.htm  
Other reports on chronic (late diagnosed) or persistent (post antibiotic treatment relapse) borreliosis documented by positive culture, microscopy and/or PCR: http://lymerick.net/persistent-borreliosis.htm

Culture in commercially available BSK-H medium* is often successfull, especially when there are a few visible spirochetes in the wet drop sample from a fresh buffy-coat prep.; after positive culture much more material can be examined by other methods, i.e. there is a possibility of PCR typing of the Borrelia strain when there is enough spirochetes. DON'T THROW MATERIAL OUT AFTER ONE MONTH as some researchers do, according to published reports on culture. In adverse conditions it may take much longer  for slow Borrelia to grow in vitro: 
http://lymerick.net/Borrelia-growth-optimum.html
*) Sigma-Aldrich http://www.sigmaaldrich.com/catalog/product/sigma/b3528?lang=en&region=DK

I read (but forgot in which ref.?) that some found that Borrelia cyst may not grow spirochetes well, unless they have experienced a colder period, like they would have experienced in a natural life situation, when residing in dormancy in a tick during the winter-time. Can the microbes sense the seasons somehow?
Many chronic patients (that are not in antibiotic treatment), descibe getting longer worse periods, shifting to a weekly relapse pattern during spring-time (late march-mid june) and during autumn (mid-august to late november), while they shift to a monthly relapse cycle during high summer (high temperature, low moisture so ticks are not questing), and monthly cycle during winter time ...
- can tickborne microbes sense somehow the seasons when ticks are active questing for blood meal out in the nature, and become active then, while they go into dormancy at times when the chance of a tick biting it host is lower?  

Please note, that all microbes that use the TICK - with ½-1 year between blood meals - as vector, ALL have the ability to go dormant in the tick for at least six to twelve months, otherwise they would not be able to keep the infectious tick-mammal-tick life cycle going. The microbes also go dormant inside their mammal host, when the environment becomes unsuitable for the spirochete form, like when the patient forms lots of antibodies, under antibiotic treatment, lack of nutrition, wrong pH, too high oxygen etc; persisters can survive common treatment and some cnb awaken later when the environment again favours regrowth of spirochetes, that has been dormant for a very long period.
This was observed by Hampp already before 1950! - http://lymerick.net/1948-Hampp.htm   

"In our experience, it is still possible to detect the pathogenic bacteria if there are less than 10 bacteria in a milliliter of centrifuged native blood samples. In comparison, the threshold for the detection of Lyme borreliosis with PCR, which is currently considered the most sensitive but can only be done in specially equipped laboratories, is between 40 and 100 germs per ml; besides, as many as possible primers specific to different sub-strains should be available."

[20 drops per ml, i.e. 1 drop is 50 microliter, i.e. there may be one spirochete per 2 drops of HIGHLY CONCENTRATED BLOOD sample, after double centrifugation! .. of course one will need to examine more drops in order to find the spirochete, if there are so few - the investigator must be very meticulous!]

http://www.ncbi.nlm.nih.gov/pubmed/22429769
The order Piroplasmida consists of several genera of tick-borne parasites that infect mammals, and to a lesser extent birds, and are therefore of medical and economic importance. Despite their importance, considerable confusion exists concerning the relationship among piroplasmid species, specifically concerning the number of genera and the intergeneric relationships.
To examine evolutionary relationships among piroplasmids, we conducted phylogenetic analyses of 192 18S rDNA sequences from the genera Theileria, Babesia and Cytauxzoon.
Our analyses revealed eight clades potentially representing distinct genera, and we distinguish the Duncani Group and Microti Group as genetically distinct groups of species requiring detailed analysis of morphology and life-history to allow formal generic description.
The piroplasmid phylogeny revealed considerable host diversity and limited host specificity, suggesting piroplasmids have undergone frequent host switches during their evolution. Our analyses provide the first reported evolutionary timescale for piroplasmids independent of the assumption of parasite-host cospeciation, which is invalid for piroplasmids.
Evolutionary rate analyses revealed considerable substitution rate heterogeneity, which we attribute to host switching and diversification. Finally, we call for a comprehensive phylogenetic, morphological and life-history analysis for these medically relevant taxa to resolve relationships and understand host specificity.

http://www.ncbi.nlm.nih.gov/pubmed/4138127     
http://wwwnc.cdc.gov/eid/article/17/1/pdfs/10-0737.pdf

"Our cases highlight that, in Europe, babesiosis can occur in healthy persons and manifest as moderate illness. The rarity of other reported cases in nonimmunocompromised patients in Europe may be related to the difficulty of diagnosing babesiosis. A stained thin blood smear is rarely performed in Europe after tick bite in healthy patients. The difficulty of detecting intra-erythrocytic forms of babesia coupled with frequent low levels of parasitemia, may result in accurate diagnoses, although acridine orange and fluorescent microscopy may assist in the detection of parasites (1). Other diagnostic tests, such as PCR and serologic analysis, are not routinely performed in France and require a reference laboratory (8). ....
Babesiosis, although difficult to diagnose, needs to be diagnosed for various reasons:
1) without treatment, babesiosis can lead to severe illness;
2) the disease can persist for a long period without symptoms, which could lead to posttransfusion cases (12); and
3) effective specific treatments are available (atovaquone plus azithromycin, or for severe cases, clindamycin and quinine) (2).
These drugs are not usually prescribed in febrile tick-bite cases; doxycycline is the usual drug used to treat tick-borne bacterial diseases. Moreover, patients with moderate infection could benefit from an atovaquone plus azithromycin regimen, which is better tolerated (13). ...
In Europe, babesiosis is probably underdiagnosed; thus, we suggest that when patients have influenza-like or malaria-like syndromes after confirmed or suspected tick bites, a blood smear be performed regardless of whether the patient is immunocompromised. Blood smear can identify not only Babesia spp. infection but also Anaplasma spp. infection, another emerging and underdiagnosed tick-borne illness. In cases of new European Babesia spp. infections, a deeper characterization of the strains by erythrocytes cultures and standardized PCR, as well as a systematic study of the patients’ immune systems, should be undertaken to enable a better understanding of this disease."

"Examination by microscopy and PCR failed to detect the parasite in the donor's blood obtained 8 months after the donation of the blood that was transfused. However, we were able to isolate Babesia parasites by inoculating the blood sample into SCID mice whose circulating red blood cells (RBCs) had been replaced with human RBCs."

"Subsequent testing of serum specimens from both [Babesia EU1] patients showed IFA reactivity to B. divergens but not to B. microti antigens; serum from the Italian patient was also tested for reactivity to WA1 antigens and was negative."

Patient characteristics table:
(CDC unfortunately had no luck in producing infection in jirds by the experimental inoculation of patients blood, so the IFA was done with B. divergens as test  antigen!)
The italian patient: "Titers of 1:64 (specimen from October 28, 1998) and 1:256 (February 15, 1999) in testing at both CDC and the Clinical Institute of Hygiene of the University of Vienna"
The Austrian patient: "Titers of 1:256 (July 31, 2000) and 1:1,024 (August 8, 2000) in testing at CDC and titers of 1:64 (July 31) and 1:1,000 (August 8) in testing at the Clinical Institute of Hygiene of the University of Vienna"

For comparison: The danish patients titer to unknown test antigen was 1:125 ...

http://www.ncbi.nlm.nih.gov/pubmed/17979540

http://www.ncbi.nlm.nih.gov/pubmed/21529420    http://wwwnc.cdc.gov/eid/article/17/5/10-1834_article.htm

"All blood and tissue samples from each organ tested were positive only for Babesia sp. EU1. ... 
Of I. ricinus ticks from the Netherlands, ≈1% are infected with Babesia sp. EU1 (6). The only confirmed reservoir host of Babesia sp. EU1 is roe deer (Capreolus capreolus) (9). The infected forest reindeer resided in a zoo in an area without direct contact with roe deer, although roe deer are abundant in the forests surrounding the zoo."  

http://www.ncbi.nlm.nih.gov/pubmed/21186774     http://www.aaem.pl/pdf/17309.pdf

"The co-infection rate of these pathogens was found to be rather low (Borrelia spp./Anaplasma phagocytophilum – 4.2%, 1/24; Borrelia spp./Babesia spp. – 4.2%, 1/24). However, due to the increased prevalence of Borrelia spp. in Ixodes ricinus ticks in Poland and the recent emergence of new tick-borne infections, it is necessary to carefully evaluate the true risk of human infection with several pathogens using more sensitive and reliable diagnostic tools.
This is the first report of human infection with Babesia spp. in Poland that has been confirmed by molecular techniques with homology of 98.9% to B. divergens or Babesia EU1."

Excerpts:
"Three recently described human cases identified in Italy, Austria and Germany were caused by infection with a species named EU1, exhibiting molecular characteristics distinct
from those of B. divergens [16, 18]. The rodent species B. microti is also present throughout Europe, although there has only been one verified case of human babesiosis due to infection with this species [20]. In recent years, co-infections of humans acquired from Ixodes ricinus ticks have been observed quite frequently in the United States [1, 4, 38] and in Europe [24, 44]. However, only three such cases have been reported so far in Poland [12, 17, 27].
In this retrospective study we examined Borrelia-seropositive individuals from southeastern Poland, where I. ricinus ticks are highly endemic, for coinfection with A. phagocytophilum and Babesia species."
"Examination of a smear of the Babesia-positive blood sample stained with Giemsa revealed Babesia spp. infection with a very low level parasitemia of 0.02% (Fig. 3)." ...

"Human co-infections with the pathogens Borrelia spp. and A. phagocytophilum with clinically (erythema migrans) and serologically (seroconversion) confirmed Lyme borreliosis as well as asymptomatic anaplasmosis (positive serology or PCR for A. phagocytophilum) have been described in Poland [12, 17] and other countries [23, 25]. Nevertheless, the few previous reports of such co-infections indicate that the resulting disease is more severe and prolonged [39*]. Since the late 1950s, two species of Babesia, B. divergens from cattle in Europe and B. microti from rodents in the northeastern and upper midwestern parts of the USA, have been shown to infect humans [26]. The species B. microti, B. divergens, B. odocoilei-like, and Babesia EU1 are known to be prevalent in I. ricinus ticks across Europe, including Poland [9, 15, 33, 43]."  [no clinical case description]

[39*] = Coinfections acquired from ixodes ticks. 2006 PDF  ..
NB In this review I found more wrong statements that has been disproven, so read carefully and check all statements, that may now be out of date!
For instance the authors claim that ONLY Bbss, B. afzelii and B. garinii are known to be human pathogenic species.
However, cases of other Borrelia burdorferi sensu lato had already been detected and reported before publication of the review:
Borrelia bisettii (Strle F, Slovenia 1999 PM "Thus far four Borrelia species were found by isolation to cause disease in humans: B. afzelii, B. garinii, B. burgdorferi sensu stricto, and B. bissettii.")
B. lusitaniae, Portugal 2004 PDF    Borrelia valaisiana Greece, 2004 HTM   Borrelia spielmanii (so far only detected in early Lyme disease AFAIK?, i.e. in EM rash) 2005 PDF
- so the authors were clearly not fully up-to-date with the published literature (or were ignorant?) when they wrote that statement in 2006! 

Proving causation is not possible after 10 years of chronic illness, as in the cases with B. lusitaniae and B. valaisiana, however both these patients were described as clinically compatible with what some of us (ILADS stance) call "chronic Lyme disease", while other colleagues (of IDSA stance) stubbornly deny the existence of persistent borrelia infection in "chronic Lyme disease", where they usually mean  "typical NEURO-borreliosis" (ass. w/ B. garinii infection), despite there are lots of culture, PCR and/or microscopy verified LATE and POST-TREATMENT cases that has been described since 1989; because the IDSA chronic Lyme denialists usually do not reference any of the published works, that speak against their stance (ignorance seem to fluorish among them), I made a list of references - persistent-borreliosis - and put it on my website in 2003, to make it easier for myself and others to check up on which references are MISSING / not discussed in articles authored by folks of IDSA stance ;)

A more recent review over Lyme borrelia diversity is discussed by Stanek (EUCALB, usually stick close to the IDSA stance in US) in "The expanding Lyme Borrelia complex—clinical significance of genomic species?" (1911) PDF ..  he still claim that only the 3 old wellknown Borrelia genospecies known from 1992 are with certainty humane pathogenic, about the others he describe them as "single" or "a few case reports", like on page 491, he writes "the presence of B. valaisiana [in neuroborreliosis] was confirmed in three cases [1]."
So I wonder, HOW many cases does it take for them to accept that Borrelia infection sometimes can be a chronic persistent perhaps life-long infection, especially in some susceptible individuals?

The recently published NIH-primate study by Embers et al. (Full text + PDF) demonstrate long term persistence of Borrelia despite 3 months ceftriaxone treatment (error in drug was corrected), this has not yet been added to my persistent borreliosis reference list!

As long as genotyping it not yet generally avaiable to us for diagnosis we will never know with certainty what type of Borrelia(s) the patient got infected with!
We need access to culture for Borrelia (at least in cases that do not get well on standard tretment for Borrelia) as well as co-infections in cases suspect of chronic late or persistent Lyme borreliose, where antigen specific diagnosis of Borrelia will support necessity for treatment and aid in choice of treatment.

MY VIEWPOINT IS:
Up until now so far 18 named genospecies have been placed in the Borrelia burgdorferi sensu lato (Bbsl) spp. complex, and so far 8 of these have been detected in material from human Borreliosis cases from Europe since 2004! - the enlarging spectrum show us that other strains of borrelia than the 3 old one, may eventually turn out be humane pathogenic, could perhaps not be detected before the PCR era, because these strains are so antigenically different, that the commonly used commercial Borrelia antibody tests, that are based on antigens from the 3 old types of Borrelia burgdorferi sensu lato, may not detect antibodies directed against the new types; more of the cases with other types have shown to been seronegative or very low seropositive, despite they harbour Borrelia!
Previously detectable Lyme antibodies have been shown to disappear, despite culture verified persistence of Borrelia in ligamentous tissue (Häupl et al. and more) ...

During very active Lyme borreliosis with detectable spirochetes by simple microscopy, many patients may test seronegative in same plasma fraction, which can be explained by immunecomplex bound antibodies are not being measured: http://kroun.ulmarweb.dk/Borrelia-IC.html (in danish, but reference mentioned are in english) ... some of them will "seroconvert" 1-2 month into clinically effective antibiotic treatment, when there is no longer antigens in blood as often (shift to monthly relapse cycle); when treatment is stopped some of them relapses again, and again spirochetes can be detected, and they may turn seronegative .. retreatment usually works and they may "seroconvert" again, when the cycle shift to monthly  .. UNDERSTANDING THE DYNAMICS OF TEST RESULTS IN RELAPSING INFECTION IS A NECESSITY! 

Because IDSA base the very diagnosis of Lyme borreliosis primarily on that the patient MUST HAVE POSITIVE SEROLOGY, two-tier, because they have chosen to use the CDC EPIDEMIC SURVEILLANCE CRITERIA as DIAGNOSTIC CRITERIA for Lyme borreliosis in the USA, despite CDC actually have warned against it! - this BLOCK for openminded discussion on the many cases of proven persistent and othen seronegative borreliosis cases. They have chosen to define"Lyme disease" in a certain way, not accepting a broader perspective; most of the controversies arises from the fact that some of the lead authors in IDSA on Borrelia have personal conflicting economic interest in serology tests!

From my viewpoint I talk about "Borrelia infection" in a much broader sence, i.e. the patient is infected, but not necessarily clinically ill, when ANY borrelia spp., broad defined i.e. including TBRF and LBRF sub-spp., can be detected by direct test methods like culture, PCR and/or microscopy (plus the patient has history indicative of borrelia like known tickbites, EM rashes, previously positive serology test) ...
Borrelia infection (antigen present and able to revert and cause symptoms, but is currently an inactive infection) may be latent, asymptomatic for weeks, months to even many years, just like in tertiary syfilis, there may be a potential risk of later relapse, in case of immune depression; such have been described in persistent borreliosis list, there are lots of published reports that supports this, really!

The KEY POINT in diagnosis of ACTIVE INFECTION is EASY, SIMPLE namely the RE-OCCURRENCE OF THE CYCLICAL CLINICAL RELAPSE PATTERN, WHICH ALLOWS US TO DETECT SPIROCHETES IN BLOOD BY MICROSCOPY DURING THE FIRST DAY OF EVERY ACTIVE FLARE PERIOD! .. in blood taken at other times spirochetes are usually not detectable!
If the patient has a "weekly" relapse pattern spirochetes can be detected in 1 per 10 days of the cycle (10% chance of lucky catch, while the risk of missing the catch is 90%); in patients on a monthly cycle 1 per 30 days, the chance of lucky catch is 3%, while the chance of missing the catch is 97%) if the clinician does NOT time the sampling for direct detection to happen day one of a new flare!
The antigen in my project cases reacted with added Borrelia antibody that had been absorbed for TBRF species and tested by the producer to be specific for Borrelia burgdorferi sensu lato (KPL).

First when we know which strain the patients have got and can compare to clinical status, it will become possible to evaluate the clinical significance of the different strains of Lyme borreliae!

Let us spare the discussion now and agree that we do not yet have information enough to make a definite conclusion on this issue, that would serve the sick best to keep an open mind to what may be possible, I think!
- there may be strains out there that has not even been found yet ...

The authors [of 39] also claim that larvae of Ixodes is not transovarially infected with Borrelia. However I know more studies proving that if the mother tick is systemically infected with Borrelia (which from 5-30% have been found to be) the Borrelia infection will be transmitted to 100% of eggs and the progeny larvae will be born infected (for instance old works by Burgdorfer himself, I have not made a list yet, is on my to-do-list)! ... More PCR studies on unfed Ixodes larvae have shown that 5-10% may harbour Borrelia spp. and therefore is potentially contagious!

There may be more disproven out-of-date or skewed statements in articles depending on "stance" of the authors, so read articles with great caution! .. be very thorough and check reference-list, not only if they reference correctly, but also which articles are missing, that they should have referenced and discussed in their context, really!

http://www.ncbi.nlm.nih.gov/pubmed/21762494     http://www.parasitesandvectors.com/content/pdf/1756-3305-4-135.pdf
"The following prevalences were detected:
Babesia spp.: 0.4% (n = 17, including one pool of two larvae) in 2009 and 0.5 to 0.7% (n = 11, including one pool of five larvae) in 2010;
Rickettsia spp.: 6.4 to 7.7% (n = 285, including 16 pools of 76 larvae) in 2009.
DNA of Bartonella spp. in I. ricinus in Bavarian public parks could not be identified.
Sequence analysis revealed the following species: Babesia sp. EU1 (n = 25), B. divergens (n = 1), B. divergens/capreoli (n = 1), B. gibsoni-like (n = 1), R. helvetica (n = 272), R. monacensis IrR/Munich (n = 12) and unspecified R. monacensis (n = 1).
The majority of coinfections were R. helvetica with A. phagocytophilum (n = 27), but coinfections between Babesia spp. and A. phagocytophilum, or Babesia spp. and R. helvetica were also detected."

=> Ratio of Babesia venatorum (EU1):Babesia divergens found in the Bavarian park ticks was 25:1 
i.e. humans and pets visiting that city park is at 25 times relative higher risk of getting infection with Babesia EU1 compared to Babesia divergens, that is, if they get a Babesia infection, since less than 1% of the examined ticks were found infected with Babesia species the general risk of aquiring Babesia infection is still quite low!

http://www.ncbi.nlm.nih.gov/pubmed/16841874    http://www.aaem.pl/pdf/13065.pdf
"The serological assays presently available are not able to discriminate between EU1 and B. divergens infections [14], hence all the cases reported to date have been ascribed to B. divergens."
"We have described a new PCR method based on the 18S rRNA gene that allows the detection of Babesia in I. ricinus ticks and in blood samples. The amplified fragments show an important number of mutations. In addition, the variability of the target fragments (25.3%) is similar to the variability of the complete gene sequences (20.3%). Because of this variability, the short amplicon of 411-452 bp of the 18S rRNA gene may be used for accurate species resolution. Indeed, we could differentiate all the Babesia species we considered (Fig. 2). Moreover, in the B. canis cluster, we clearly could separate the two subspecies B. canis vogeli and B. canis canis (Fig. 2). The analytical sensitivity of the PCR assays has been shown to be 27.5 fg/reaction of B. divergens DNA. However, the majority of the preview reports describing PCR for the detection of Babesia in ticks did not report the sensitivity, with the exception of a study which showed a limit of detection of 50 organisms/ml of canine
whole blood [3]. ...
This is the first study reporting the presence of Babesia sp. EU1 in Switzerland. This organism was detected in ticks collected in two areas located North (Neuchâtel) and South of the Alps (Ticino). The presence in Switzerland of B. microti and B. divergens was also confirmed. ...
Recently, Meer-Scherrer et al. [20] described the firsthuman case due to B. microti in a Swiss patient. However, the behaviour and the virulence of the European B. microti strains might be different from those of North America. Interestingly, in another study, it has been suggested that B. microti in the United States may be more virulent for humans than the B. microti-like isolated in Japan [25]. It is important to consider that B. microti actually consists of a genetical species complex based on the 18S rRNA gene: Babesia isolated in rodents divide in two clades, one zoonotic, including our samples, and the other maintained only in rodents [11].
Recently, two cases of human babesiosis, in Italy and Austria, have been attributed to EU1 [14]. Moreover, in Slovenia ticks have been found infected by this species [6]. The EU1 sequences obtained in these different countries, including Switzerland, are identical or very close (99.8% of similarity) and thus no specific geographic association may be identified. An important issue concerning Babesia sp. EU1 is to determine whether it is an emergent species."

http://www.ncbi.nlm.nih.gov/pubmed/20575647    

"We report the first molecular evidence of the presence of Babesia sp. EU1 and Babesia microti in Ixodes ricinus ticks in Belgium. A 1-year national survey collected 1005 ticks from cats and dogs. A polymerase chain reaction technique amplifying a part of the 18S rRNA gene detected Babesia spp. in 11 out of 841 selected and validated tick extracts.
Subsequent sequencing identified Ba. microti (n=3) and Babesia sp. EU1 (n=6). This study has demonstrated a low infection rate (1.31% with 95% CI: 0.65-2.33) of Babesia spp. carriage in I. ricinus ticks in Belgium but, for the first time, reports two potentially zoonotic species belonging to this genus. Coinfection with Ba. microti and Borrelia burgdorferi sensu stricto also was demonstrated. In addition, this study clearly demonstrates that inhibitors of polymerase chain reaction amplification are present in engorged ticks."

http://www.ncbi.nlm.nih.gov/pubmed/21158500

"...  Pathogen prevalence rates were evaluated by polymerase chain reaction detection and sequencing in questing ticks, individually for adults and in pools of 10 for nymphs. In addition to finding micro-organisms corresponding to symbionts, we found high prevalence rates of B. burgdorferi s.l. (32% of adult females and 10% of nymphs)low to moderate ones of Anaplasma phagocytophilum (~1%), spotted fever group Rickettsia spp. (~6%), Babesia sp. EU1 (~1%), Bartonella birtlesii (0.1%), and Francisella tularensis (!1%).
Our findings extend the knowledge of the geographical distribution of these endemic and emergent pathogens and support the conclusion that ticks are important vectors of pathogenic micro-organisms in suburban forests. Moreover, tick coinfection with multiple pathogens was found to occur frequently, which poses a serious challenge for diagnosis and appropriate treatment. The incrimination of these pathogens in potentially severe pathologies requires widespread surveillance to assess the risk of infection, thereby facilitating diagnosis and treatment, as well as raising local awareness of tick-borne diseases." and

http://www.ncbi.nlm.nih.gov/pubmed/21395407

"The presence of Babesia spp. was studied in 2603 Ixodes ricinus and Ixodes persulcatus ticks collected at seven sites in Estonia. By reverse line blot screening, spp. was detected in 36 (1.4%) ticks, among them 18 (0.7%) were further recognized by a BabesiaBabesia microti probe, 3 (0.1%) by a Babesia divergens probe, and the other 15 (0.6%) were recognized only by the universal Babesia spp. "catch all" probe. Sequence analyses of 6 of these 15 samples revealed that all of them belonged to Babesia sp. EU1. B. microti was detected in both tick species I. ricinus and I. persulcatus at the seven sites, whereas B. divergens-like and Babesia sp. EU1Babesia sp. EU1 share a high rate of similarity and are closely related to sequences from other European countries, Siberia, and United States. The present study demonstrated for the first time the existence and distribution of Babesia spp. in I. persulcatus and I. ricinus ticks in Estonia."

http://www.ncbi.nlm.nih.gov/pubmed/21395425

http://www.ncbi.nlm.nih.gov/pubmed/21417929

http://www.ncbi.nlm.nih.gov/pubmed/22452970      http://www.parasitesandvectors.com/content/pdf/1756-3305-5-61.pdf

"Eleven pathogens were detected in 77 out of 193 ticks collected in 14 sites. The most common microorganisms detected were Borrelia burgdorferi sensu lato (17.6%), Rickettsia helvetica (13.1%), and "Ca. N. [N= Neoehrlichia] mikurensis" (10.5%).
Within the B. burgdorferi complex, four genotypes (i.e., B. valaisiana, B. garinii, B. afzelii, and B. burgdorferi sensu stricto) were found. Less prevalent pathogens included R. monacensis (3.7%), TBE virus (2.1%), A. phagocytophilum (1.5%), Bartonella spp. (1%), and Babesia EU1 (0.5%).
Co-infections by more than one pathogen were diagnosed in 22% of infected ticks." 

http://www.ncbi.nlm.nih.gov/pubmed/22493116    

"In a region-wide serologic study carried out in 2004 on free-ranging hunted roe deer in various landscapes, we found that 58% of the animals (237 out of 406) were antibody positive for Babesia divergens antigen. Serologic and infection status was also analyzed for 327 roe deer live-trapped in two fenced forest areas over 5 yr (2004-08). For two consecutive years during this period, 92 and 94% of the deer in these closed populations were antibody-positive for B. divergens.
Babesia spp. were isolated in autologous red blood cell culture for 131 of the trapped animals (40%).
Molecular typing was done on 76 isolates with polymerase chain reaction (PCR)-restriction fragment length polymorphism methods targeted at the 18S ribosomal subunit gene (18 isolates) and the Bd37 gene coding for a merozoïte surface antigen implicated in a protective response (60 isolates).
Results indicated continuous cocirculation of B. capreoli and B. venatorum in both forests and possible coinfection of animals with both species. No infection with B. divergens was detected. Fifteen isolates were confirmed to be B. capreoli by sequencing part of the 18S rRNA gene. Using PCR detection of the Bd37 gene, all nine isolates of B. venatorum in this study were negative, whereas the 15 confirmed and 50 putative B. capreoli isolates showed very variable restriction profiles, distinct from those known for Bd37 in B. divergens. Two isolates showed conflicting results, suggestive of mixed infection."

"Two of the patients with elevated titers to WA1 had a prolonged disease course, one with elevated liver enzymes."

http://www.ncbi.nlm.nih.gov/pubmed/22168221    
Molecular and indirect fluorescent antibody (IFA) analyses were negative for B. microti but were positive for B. duncani (IFA titer, 1:1024).
The complete 18S ribosomal RNA gene of the parasite was amplified from a blood specimen; the DNA sequence was identical to the sequence for the index WA1 parasite isolated in 1991.
The patient's case prompted a transfusion investigation: 34 of 38 pertinent blood donors were evaluated, none of whom tested positive by B. microti IFA.
The implicated donor - a 67-year-old California resident - had a B. duncani titer of 1:4096; B. duncani also was isolated by inoculating jirds (Mongolian gerbils) with a blood specimen from March 2009, more than 10 months after his index donation in April 2008. The patient's case was diagnosed more than 4 months after the implicated transfusion in May 2008.

THIS IS SO INSTRUCTIVE that I do not find it necessary to list other reports that likewise show that
commonly used assays for B. microti does not detect infection with B. duncani (WA1)

It shows the timing, long persistence of babesia if not treated, risk for blood recipient if the asymptomatic low level carrier decides to donate blood. 
There are no screening for Babesia, blood banks just ask if the patient feel healthy, do not exclude previous borrelia / tickbitten as donors, do not apply any tests for Babesia on donors blood. 
The Japanese donor transmitted case - http://lymerick.net/York2004/Japanese-donor-Babesia.htm    http://www.ncbi.nlm.nih.gov/pubmed?term=babesi*+Japan+donor -
http://jcm.asm.org/content/38/12/4511.full.pdf
"The donor's serum exhibited a high antibody titer against the isolate from the patient, whereas it exhibited only a weak cross-reaction against B. microti strains isolated in the United States."

Indirect fluorescent-antibody test (IFAT). An approximately 50% suspension of infected RBCs which had 30 to 50% parasitemia was prepared in phosphatebuffered
saline (PBS; pH 7.2) containing 50% fetal bovine serum. Roughly 0.3-ml  aliquots were spread into each well of a 24-well HT Coating Slide (MS 342 BL; Bokusui Brown, Tokyo, Japan) and were then dried. The slides were fixed in acetone for 5 min and were then immediately transferred into PBS to lyse the RBCs. Following removal of solution by briefly blotting with a filter paper, the slides were placed in a moisturized chamber, and 15 ml of serial twofold dilutions of serum specimens, starting from 1:25, was added to each well. After 1 h of incubation at room temperature, the slides were washed in PBS, and 15 ml of fluorescein isothiocyanate-labeled protein A (EY Laboratories, Inc., San Mateo, Calif.) diluted 1:200 in 5% fetal bovine serum–PBS was added to each well. The slides were incubated at room temperature for 1 h and washed in PBS. Component A of the Slowfade antifade kit (Molecular Probes, Eugene, Oreg.) was mounted onto each well, and cover glasses were placed on the slides. Fluorescent parasites in RBCs were observed with a fluorescence microscope at a magnification of 3200.
Reference strains of B. microti. The Gray strain (4) was a gift from J. Dickerson, Division of Parasitic Diseases, Centers for Disease Control and Prevention.
Strain Gray-Mo, a mouse-adapted substrain of the Gray strain, has been described previously (15). The GI and AJ strains were provided by H. Saeki, Nippon Veterinary and Animal Science University. Syrian hamsters were used for propagation of the Gray strain, and the antibodies in their convalescentphase sera were used as the specific antibodies. The Gray-Mo, GI and AJ strains were propagated in C.B-17 scid mice, and antisera against these strains were prepared with BALB/c mice.

- shows another problem, that there may be local variants in different places of the world, where strains of genetically similar strains, differ enough from each other  in surface antigens, to not be readily detected by a B. microti anitbody test based on a strain from the USA!
=> test should be done with local microbe strains! 

http://wwwnc.cdc.gov/eid/article/10/4/03-0377_article.htm
Most reported U.S. zoonotic cases of babesiosis have occurred in the Northeast and been caused by Babesia microti.
In Washington State, three cases of babesiosis have been reported previously, which were caused by WA1 (for “Washington 1”)-type parasites.
We investigated a case of babesiosis in Washington in an 82–year-old man whose spleen had been removed and whose parasitemia level was 41.4%.
The complete 18S ribosomal RNA gene of the parasite was amplified from specimens of his whole blood by polymerase chain reaction.
Phylogenetic analysis showed the parasite is most closely related, but not identical, to B. divergens (similarity score, 99.5%), a bovine parasite in Europe.
By indirect fluorescent-antibody testing, his serum reacted to B. divergens but not to B. microti or WA1 antigens.
This case demonstrates that babesiosis can be caused by novel parasites detectable by manual examination of blood smears but not by serologic or molecular testing for B. microti or WA1-type parasites.

THE KEY POINT!  ...  
Neg. antibody test and negative PCR test can not be used to rule out Babesia infections, particularly NOT when ringforms can be detected by simple  microscopy, which still remains "the gold standard" test! 
Negative thin and thick blood smears can not be used to rule o

"Nowadays, portable fluorescent microscopes usinglight emitting diode (LED) technology, and pre-prepared glass slides with fluorescent reagent to label parasites, are available commercially [38]. Although the QBC technique is simple, reliable, and user-friendly, it requires specialized instrumentation, is more costly than conventional light microscopy, and is poor at determining species and numbers of parasites."

38: Partec reagents and accessories.  http://www.partec.com   http://www.partec.com/reagents-accessories/essential-healthcare/reagent-kits.html
05-8951 Partec Rapid Malaria Test, 200 tests
Ready-prepared and ready-to-use Malaria test slides with dried-in reagents for easy-to-perform 1-step protocol and immediate analysis. Maximum shelf life: 12 months.